mTOR translates microenvironmental signals into cell differentiation responses and controls cell fate including authophagy and apoptosis. Phenotypic conversion of mesenchymal stem cells (MSC) towards osteoblast lineages is instrumental in adverse vascular remodeling and transplant arteriosclerosis (TA). We reasoned distinct involvement of mTOR complex (mTORC) 1 and mTORC2 in regulation of MSC regenerative capacity.

Human MSC in calcification medium were stimulated with growth factors implicated in TA in presence or absence of a pharmacologic mTOR inhibitor (rapa). Osteoblastic differentiation, induction of mTORC1 and mTORC2 signaling effectors, proliferation, apoptosis and autophagy were determined. Lentiviral gene transfer served to define decisive roles of different signaling elements.

b-FGF and PDGF-BB enhanced calcification and osteoblastic differentiation which was precluded by TGF-Β1. CTGF and FGF-23 did not modulate the response to calcifying medium. Rapa strongly inhibited pro-arteriosclerotic transformation in response to growth factors in a dose dependent manner. Rapa also reduced mTORC1 activity which lead to decreased proliferation and increased autophagosome related LC3. In parallel, mTORC2 signaling was enhanced and linked to anti-apoptotic effects. Abrogation of mTORC2 function by lentiviral transfer of shRNA targeting the mTORC2 defining component rictor reversed the protective effect of rapa on cell differentiation by means of induction of apoptosis and increased extracellular calcium content.

Our findings define selective roles for mTORC1 and mTORC2 in MSC during pharmacologic mTOR targeting. Inhibition of mTORC1 activity and simultaneous enhancement of mTORC2 related responses were instrumental in inhibition of apoptosis, induction of authophagy and control of extracellular calcium contents. Functional mTORC2 signaling was requisite for the maintenance of these protective cell programs. We provide a new mechanistic link between mTORC2 activation via mTORC1 inhibition which may boost endogenous regenerative potential of MSC. Strategies aiming to enhance mTORC2 activity could help to reduce TA after solid organ transplantation.

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